Effects of ABCA1 SNPs, including the C-105T novel variant, on serum lipids of Brazilian individuals

Results Linkage disequilibrium was found between the SNPs C-105T and C-14T in the HC group. HC individuals carrying -105CT/TT genotypes had higher serum HDL-c and lower triglyceride and VLDL-c concentrations as well as lower TG/HDL-c ratio compared to the -105CC carriers ( p < 0.05). The R219K SNP was associated with reduced serum triglyceride, VLDL-c and TG/HDL-c ratio in the HC group ( p < 0.05), and with an increased serum apoAI in NL individuals. The effects of ABCA1 SNPs on basal serum lipids of HC individuals were not modified by atorvastatin treatment. Conclusions The ABCA1 SNPs R219K and C-105T were associated with a less atherogenic lipid profile but not with the lowering-cholesterol response to atorvastatin in a Brazilian population. Keywords ABCA1 Pharmacogenetics Plasma lipids SNPs, Statins 1 Introduction The association between high levels of high-density lipoprotein cholesterol (HDL-c) and a decreased risk for cardiovascular disease has been well established through epidemiological and clinical studies [1–3] . This relationship is supported by the potential antiatherogenic properties of HDL, including its involvement on reverse cholesterol transport (RCT), in which cholesterol from peripheral tissues, including foam cells of the arterial wall, is returned to the liver for elimination through the biliar system [4] . The ATP-binding cassette transporter A1 (ABCA1) has an essential role on regulation of HDL metabolism [5,6] and there is strong evidence that ABCA1 is also a key regulator of RCT [7] . This protein mediates the transport of cholesterol, phospholipids, and other lipophilic molecules across cellular membranes, where they are removed from cells by lipid-poor HDL apolipoproteins [8] . In fact, ABCA1-mediated assembly of phospholipid and free cholesterol with apolipoprotein A-I plays an important role in HDL biogenesis [9,10] . Mutations in the ABCA1 gene cause Tangier disease and familial hypoalphalipoproteinemia disorders characterized by deficiency in cholesterol efflux from cells and reduced HDL-c in serum, as well as an increased risk for atherosclerosis [11–15] . In addition, many common variants of ABCA1 have been associated with a wide range of clinical and biochemical phenotypes including the variations on HDL-c serum levels [16,17] . The R219K single nucleotide polymorphism (SNP) has been associated with protection against coronary artery disease (CAD) risk and severity [18–23] . This protective effect appears to be related to an increased HDL-c and/or reduced triglyceride levels [18,21,24,25] , but these results were not reproduced in all studies [19,20,26] . Other SNPs located in the promoter region of ABCA1 , such as C-14T (also described as C69T), were associated with the severity of atherosclerosis and increased risk for CAD apparently without changing the plasma lipid levels [16,17,27] . It is well known that treatment with HMGCoA reductase inhibitors, known as statins, reduce the relative risk of coronary events, primarily by lowering the low-density lipoprotein cholesterol (LDL-c) serum levels [28] . In addition, the effects of statins on other serum lipids are highly variable and they have been associated with changes in basal concentrations of HDL-c [29,30] . The present study describes the effects of the novel variant C-105T and other SNPs of ABCA1 on serum lipid profile and on the lowering-cholesterol response to atorvastatin in a population of Brazilian individuals. 2 Materials and methods 2.1 Subjects and study protocol From 2003 to 2006, 367 unrelated individuals (112 men and 255 women, aged 29 to 81 y) were selected among the outpatients evaluated for the presence of risk factors for CAD at the Institute Dante Pazzanese of Cardiology and the University Hospital of the Sao Paulo University (Sao Paulo City, Brazil). Two-hundred twenty-four individuals had primary hypercholesterolemia (HC), as defined by the NCEP (National Cholesterol Education Program) [31] , and 143 were normolipidemic (NL) with total cholesterol up to 5.18 mmol/l and triglyceride < 1.69 mmol/l. Subjects with thyroid, liver or renal disease, diabetes, hypertriglyceridemia (triglyceride > 4.52 mmol/l) or individuals under treatment with lipid-lowering drugs, hormone replacement or oral contraceptives were excluded from the study. Information on age, height and weight, medication in use, cigarette smoking, alcohol consumption, physical activity, as well as history of menopause, hypertension, obesity, and family history of CAD was recorded. Current cigarette smoking was defined as a daily intake of one or more cigarette. We considered as “alcohol consumption” a daily intake of beer, wine, and distilled spirits of ≥ 1 g/day. Physical activity was considered the practice of sports, for example walking, running or swimming, for at least 2 h per week. Each individual declared his ethnic group during the interview. Anthropometric measurements, such as body mass index (BMI), were taken from each participant. Subjects with BMI > 30 kg/m 2 were classified as obese [32] . Individuals with systolic/diastolic blood pressure ≥ 140/90 mm Hg or under antihypertensive therapy were considered hypertensive [33] . The HC patients were oriented to follow a low fat diet according to the American Heart Association recommendation [34] during 4 weeks, in order to exclude those with diet-induced hypercholesterolemia. After this screening, those with LDL cholesterol higher than 4.14 mmol/l ( n = 141) were treated with atorvastatin (10 mg orally once-a-day) for 4 weeks. Before and after administration of atorvastatin, all patients were evaluated for serum concentrations of lipids, alanine aminotransferase (ALT) and creatine kinase (CK). Serum lipids were measured to monitor the response to atorvastatin. ALT and CK were determined in order to detect possible adverse drug reactions. The study protocol was approved by the local Ethical Committees and informed consent was obtained from each participant. 2.2 Measurement of lipid and lipoproteins Blood samples were collected after a 12-h fasting. Patients receiving atorvastatin had blood drawn prior and after the 4-week treatment. Total cholesterol, HDL-c and triglycerides (TG) were measured using routine enzymatic methods. Values of LDL-c and very low-density lipoprotein cholesterol (VLDL-c) were calculated using the Friedewald formula [35] . Plasma apolipoprotein AI (apoAI) and apolipoprotein B (apoB) were determined by nefelometry. ApoB/apoAI and TG/HDL-c ratios were also estimated [36,37] . Serum ALT and CK tests were determined by kinetic methods using the ADVIA®1650 analyzer (Siemens Medical/Bayer Diagnostics, Tarrytown, NY). 2.3 DNA extraction and ABCA1 genotyping Genomic DNA was extracted from EDTA-treated whole blood samples using a salting-out method [38] . ABCA1 C-14T and R219K SNPs were detected by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays. The primers for C-14T (forward: 5′-CTCCACGTGCTTTCTGCTGA-3′, and reverse 5′-CACTCACTCTCGCTCGCAAT-3′) and R219K (forward: 5′-GAAGAGATGATTCAACTTGGTGAC-3′, and reverse 5′-GCCCAAAAGTCTGAAAGAACAC-3′) were selected using the ABCA1 reference sequence (GenBank Acession number AF275948 ) previously described [39] . PCR assays contained 100 ng genomic DNA, 200 nmol/l primers (Invitrogen Corporation, CA, USA), 200 μmol/l dNTPs (GE Healthcare, Buckinghamshire, England), 1.0 U DNA polymerase and PCR buffer [50 mmol/l KCl, 20 mmol/l (NH 4 ) 2 SO 4 , 2 mmol/l MgCl 2 , 75 mmol/l Tris–HCl (pH 9.0)] (Biotools, Madrid, Spain) in 50 μl total volume. The PCR thermal cycling conditions consisted of initial incubation at 98 °C for 3 min, followed by 29 cycles at 94 °C for 1 min, 57 °C for 2 min and 72 °C for 1 min for C-14T polymorphism or by 32 cycles at 94 °C for 45 s, 58 °C for 1.5 min and 72 °C for 1.5 min for R219K SNP and a final extension period of 72 °C for 10 min. Amplification was carried out in a PTC-200 Thermal Cycler (MJ Research Inc., MA). PCR products of C-14T and R219K SNPs (173 bp and 295 bp respectively) were analyzed by 1% agarose gel electrophoresis after ethidium bromide staining. PCR products of the C-14T and R219K SNPs were initially digested with the MspI and StyI endonucleases (New England Biolabs Inc., Ipswich MA) respectively, at 37 °C. Restriction fragments were identified by 8% polyacrylamide gel electrophoresis after silver staining. As we analyzed the C-14T SNP, we found that some DNA samples showed an abnormal RFLP profile suggesting the presence of a novel polymorphism in the promoter region of the ABCA1 that was confirmed by DNA sequencing. This new polymorphism received the name C-105T (the submitted SNP number: NCBI dbSNP ss_76859909 ) according to the transcription starting site of the ABCA1 sequence [39] . In order to detect both the C-14T and the C-105T ABCA1 SNPs using the same PCR product, we developed new RFLP strategies using BsmAI and AlwNI endonucleases (New England Biolabs) that recognize the -14T and -105T alleles, respectively. Reaction mixtures were incubated at temperatures specified by the manufacturer and restriction fragments were identified by 8% polyacrylamide gel electrophoresis after silver staining. Identifications of the ABCA1 C-14T and C-105T genotypes by RFLP are shown in Fig. 1 . The accuracy of the genotyping was evaluated by performing duplicate analysis of 20% of the samples randomly selected. In addition, control samples containing heterozygous ABCA1 polymorphism were included in each run. 2.4 DNA sequencing Purified PCR products were sequenced in both directions using the capillary electrophoresis system MegaBACE 1000 (GE Healthcare, Buckinghamshire, U.K.) and a standard protocol for the Termo Sequenase II dye terminator cycle sequencing kit (GE Healthcare). Results from DNA sequencing are shown in Fig. 1 . 2.5 Statistical analysis Categorical variables were compared by χ 2 test or Exact Fisher test. χ 2 also was used to test Hardy–Weinberg equilibrium (HWE). To evaluate the association between ABCA1 genotype and serum lipid concentration, individuals carrying the homozygous form of the less common allele were grouped with the heterozygous carriers. Continuous variables were presented as means ± SD and the effects of the polymorphisms and haplotypes on serum lipid profile were evaluated by t -test and one-way analysis of variance (ANOVA), respectively. Variables without normal distribution were log transformed for analysis. The linkage disequilibrium (LD) between 2 SNPs was measured using the D ′ index and it was determined using SNPAnalyzer software [40] . Univariate logistic regression analysis was used to establish correlations between hypercholesterolemia and independent variables. The results of this analysis were adjusted for the covariates: age, gender, ethnics, hypertension, obesity, smoking and physical activity. Statistical tests were performed by SAS System for Windows software version 8.02 (SAS Institute Inc., 1999–2001, Cary, NC). Significance level was set at p < 0.05. 3 Results Anthropometric, demographic and clinical results of the studied group are shown in Table 1 . Seven patients did not inform their ethnic group (4 HC and 3 NL). Frequencies of gender, ethnicity, smoking, alcohol consumption and physical activity were similar between HC and NL groups ( p > 0.05). However, mean age, BMI values and frequencies of obesity, hypertension, family history of CAD or menopausal women were higher in HC group when compared with NL ( p < 0.05). As expected, concentrations of basal serum lipids were higher in HC than in NL patients ( p < 0.001), with exception of HDL-c and apoAI that were not significantly different between these 2 groups ( Table 1 ). The apoB/apoAI and TG/HDL-c ratios in NL group were lower than in HC patients ( p < 0.001). No significant variations in ALT and CK serum activities were found in patients treated with atorvastatin (data not shown). Genotype and allele frequencies of ABCA1 R219K, C-14T and C-105T SNPs in NL and HC groups are shown in Table 2 . The genotype distribution of the R219K and C-14T variants were in HWE in our sample population ( p > 0.05). On the contrary, the C-105T SNP showed deviations from HWE ( p < 0.05). Frequencies of the less common ABCA1 alleles in HC individuals (219K, 42.0%; -14T, 35.0%; -105T, 2.0%) were similar to those found in the NL group (219K, 40.6%; -14T, 33.6%; -105T, 1.4%; p > 0.05) ( Table 2 ). There were no differences in gender frequencies between polymorphism carriers and non-carriers (data not shown). In the NL group, the C-14T and C-105T SNPs were not associated with differences in basal serum lipid concentration, but 219RK/KK genotype carriers had higher apoAI ( p = 0.035) serum concentrations than the 219RR genotype carriers ( Tables 3, 4 and 5 ). In the HC group, the 219RK/KK genotype carriers had lower basal serum triglyceride ( p = 0.039) and VLDL-c ( p = 0.036), and a lower TG/HDL-c ratio ( p = 0.030) than those carrying the 219RR genotype ( Table 3 ). Likewise, the -105T allele (-105CT/TT genotypes) was associated with lower basal serum triglyceride ( p = 0.010) and VLDL-c ( p = 0.010), a lower TG/HDL-c ratio ( p = 0.001) and higher HDL-c ( p = 0.009) serum concentration ( Table 5 ). On the other hand, C-14T SNP was not associated with differences in serum lipids in hypercholesterolemic patients ( Table 4 ). These results are suggestive that ABCA1 R219K SNP and possibly the novel variant C-105T are associated with a less atherogenic serum lipid profile. A linkage disequilibrium was found between C-105T and C-14T SNPs in both NL and HC groups ( D ′ = 1.00). Haplotype analysis between C-14T and C-105T SNPs showed that in the HC group, -14CT + TT/-105CT + TT haplotype carriers had higher HDL-c level ( p = 0.017), lower serum triglyceride ( p = 0.006) and VLDL-c ( p = 0.005) and lower TG/HDL-c ratio ( p = 0.002) than those carrying other haplotypes ( Table 6 ). But, as there are few -105CT/TT genotype carriers in our study, other studies are necessary to confirm these results. Data from univariate logistic regression analysis showed that the ABCA1 SNPs or haplotypes were not associated with hypercholesterolemia in this study ( p > 0.05, data not shown). The effects of ABCA1 SNPs on serum lipid profile were also evaluated in response to atorvastatin (10 mg/day/4 weeks). After the therapy, R219K and C-105T SNPs continued to show an association with a less atherogenic serum lipid profile ( Fig. 2 ), but no ABCA1 SNP was associated with percentual variations in serum lipids after the treatment (data not shown). 4 Discussion In the present study, the frequency of the ABCA1 219K allele (NL, 40.6%; HC, 42.0%) was similar ( p > 0.05) to that found in Caucasian, Hispanic or Chinese descendent Americans (28.0–40.0%) [18] , Turks (38.5%) [41] and Pakistanis (37.3%) [26] . However, the frequencies found in our study were higher ( p < 0.05) than those found in a European population with CAD (21.0% and 25.4%) [21,22] and in white Americans (26.2%) [42] , but were lower ( p < 0.05) than in black Americans (59.5% and 64.0%) [18,42] . It is noteworthy that the rare allele R219K SNP showed a relationship with lower TG/HDL-c ratio, an indirect measurement of small dense LDL particles [37] , that has an important role in the atherosclerotic process [43] . Our findings suggest that the R219K SNP was associated with a reduced risk for CAD in this sample population. Previous studies based on case-control models have shown that the 219K allele seems to modify CAD risk regardless of important modification of plasma lipids [19,20,22] . In this study ABCA1 R219K SNP was associated with an increased serum apoAI in the NL group and with a reduced serum triglyceride and VLDL-c in HC individuals. Similar results were found in European population and in Japanese children [21,25] . The putative antiatherogenic effect of the 219K allele is not completely understood. It has been proposed that the substitution of an arginine by a lysine at residue 219 may alter the conformation of the extracellular domain of the ABCA1 protein enhancing its interaction with apoAI and increasing the efficiency of phospholipid and cholesterol transfer from intracellular stores to the plasma membrane [18] . Regarding ABCA1 C-14T SNP, the -14T allele frequencies in HC (35.0%) and NL (33.6%) groups were similar ( p > 0.05) to that found in a random U.S. population (38.0%) [44] , a random Turkish population (37.7%) [41] , and in a population of Malay, Indian and Chinese men in Singapore either healthy or with CAD (31.7 to 44.3%) [45] , but higher ( p < 0.05) compared to that observed in Dutch men with CAD (13.8%) [27] . We found no significant relationship between C-14T polymorphism and risk for CAD in our population. However, this particular SNP was associated with risk for CAD in an Indian case-control study [45] and increased number of coronary events atherosclerotic progression in Dutch men with CAD [27] . These differences may be due to the fact that in our study the risk for CAD was estimated in individuals without cardiac disease, while the other studies were performed in individuals with CAD. In addition, we found no association between the ABCA1 C-14T polymorphism and serum lipids in our population. Similar results were reported in men from Asian and European populations with or without CAD [27,45] . However, an association between -14T allele and increased serum HDL-c has been shown in Turkish and healthy Chinese men [41,45] . The rare allele of the novel C-105T SNP of ABCA1 was found in low frequencies in both HC (2.0%) and NL (1.4%) groups. Low frequencies and size of the sample may have contributed to the deviations of the C-105T genotype distribution from HWE found in our study. It is possible that other variables such as admixture and selection bias of patients also influenced genotype distributions as well [46] . Therefore, studies carried out with larger samples are necessary to confirm our results. The C-105T SNP, like R219K also appears to be related to a less atherogenic lipid profile since the -105T allele was associated with lower triglyceride and VLDL-c and higher HDL-c levels and reduced small LDL (given by the TG/HDL-c ratio) [37] in the HC group. However, these data need to be reevaluated in studies with more individuals. The mechanism underlying the changes caused by this polymorphism is still unclear. It has been noted that variations in the promoter regions of genes may potentially alter gene expression by changing the affinity or specificity of transcription factor binding to DNA and/or by altering the kinetics of transcription initiation [47] . Several transcription initiation sites have been identified for ABCA1 , and levels of groups of these transcripts have been characterized [48] . According to the starting sites described by Pullinger et al. [44] or by Schwartz et al. [49] , the C-105T SNP is located near a TATA box region. Moreover, this SNP is also located between the SP1 and AP1 motifs in the human ABCA1 promoter [39,44,50] . Langmann et al. [51] investigated the functional properties of the conserved GnC motif that bind the zinc finger protein Sp1 within the core promoter sequence. These authors have shown that the TATA box and the GnC motif (located next to the C-105T SNP) are pivotal elements for the basal transcriptional activation of ABCA1 in macrophage and liver cells. Moreover, overexpression of SP1 upregulated ABCA1 mRNA expression in HeLa cells and enhanced cellular cholesterol and phospholipid efflux in RAW 246.7 macrophages. The importance of SNPs in modifying gene expression has been demonstrated by several previous studies [52,53] . Then, it is possible that the -105C>T transition in the ABCA1 promoter modifies the mRNA expression. It could lead to changes in the cellular phospholipid and cholesterol efflux and in the HDL metabolism, that consequently would lead to alteration of the triglyceride metabolism. The negative correlation between plasma HDL-c and fasting triglyceride levels is well known [54] , however the precise mechanism regulating this correlation is yet poorly understood. It may result from an exchange in cholesterol and triglyceride between HDL and triglyceride-rich lipoproteins mediated by the cholesterol ester transfer protein (CETP) [21] . In Brazilian individuals we studied only three SNPs and we observed a linkage disequilibrium between C-14T SNP and the novel variant C-105T. In a European population the ABCA1 C-14T SNP was in linkage disequilibrium with other polymorphisms [23] and in a Turkish population, the -14TT genotype in combination with the 219KK genotype was associated with higher serum HDL-c levels [41] . Therefore, the interpretation of our findings should take in consideration possible interactions with other polymorphisms or even with environmental factors. Several genetic polymorphisms that may play a role in the different responses to the lipid-lowering therapy have been described. ABCA1 polymorphisms seem to have a minor impact in this response compared with SNPs in other genes involved in cholesterol metabolism, i.e., LDLR , APOE , APOB [55] . In our study, the HDL-c response to atorvastatin was independent of the ABCA1 C-105T, C-14T or R219K SNP. Likewise, in Dutch men treated with pravastatin there was no association between progression of atherosclerosis and C-14T SNP [27] . The authors suggested that pravastatin therapy may be able to overcome the effects of this variant in Dutch population. On the other hand, Lutucuta et al. [56] found a significant association between the ABCA1 C-477T variant and differences on serum apoAI but not on other lipids with fluvastatin treatment. In summary, C-105T SNP, a novel ABCA1 variant, is linked to the C-14T SNP. In addition, R219K and, possibly, C-105T polymorphisms are associated with a less atherogenic serum lipid profile in Brazilian hypercholesterolemic patients before and after treatment with atorvastatin. 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